Vaccine against HBV and HPV

ABSTRACT

Novel combined vaccine compositions are provided, comprising a hepatitis B viral antigen and optionally in addition one or more of the following: an EBV antigen, a hepatitis A antigen or inactivated attenuated virus, a herpes simplex viral antigen, a VZV antigen, a  Toxoplasma gondii  antigen. The vaccine compositions are formulated with an adjuvant which is a preferential stimulator of TH1 cell response such as 3D-MPL and QS21.

This application is a continuation of application Ser. No. 10/070,468,filed Oct. 1, 2002, now abandoned which is a 371 of Internationalapplication No. PCT/EP00/08728, filed 06 Sep. 2000.

This invention relates to novel vaccine formulations, methods forpreparing them and their use in therapy. In particular the presentinvention relates to combination vaccines for administration toadolescents.

Papillomavirus are small DNA tumour viruses, which are highly speciesspecific. So far, over 70 individual human papillomavirus (HPV)genotypes have been described. HPVs are generally specific either forskin (e.g. HPV-1 and-2) or mucosal surfaces (e.g. HPV-6 and-11) andusually cause benign tumours (warts) that persist for several months oryears. Such benign tumours may be distressing for the individualsconcerned but tend not to be life threatening, with a few exceptions.

Some HPVs are also associated with cancers. The strongest positiveassociation between an HPV and human cancer is that which exists betweenHPV-16 and HPV-18 and cervical carcinoma. Cervical cancer is the mostcommon malignancy in developing countries, with about 500,000 new casesoccurring in the world each year. It is now technically feasible toactively combat primary HPV-16 infections, and even establishedHPV-16-containing cancers, using vaccines. For a review on the prospectsfor prophylactic and therapeutic vaccination against HPV-16 see CasonJ., Clin. Immunother. 1994; 1(4) 293-306 and Hagenesee M. E., Infectionsin Medicine 1997 14(7) 555-556, 559-564.

Today, the different types of HPVs have been isolated and characterizedwith the help of cloning systems in bacteria and more recently by PCRamplification. The molecular organisation of the HPV genomes has beendefined on a comparative basis with that of the well characterizedbovine papillomavirus type 1 (BPV1).

Other HPV serotypes of particular interest are 31, 33 and 45.

Although minor variations do occur, all HPVs genomes described have atleast seven early genes, E1 and E7 and two late genes L1 and L2. Inaddition, an upstream regulatory region harbors the regulatory sequenceswhich appear to control most transcriptional events of the HPV genome.

E1 and E2 genes are involved in viral replication and transcriptionalcontrol, respectively and tend to be disrupted by viral integration. E6and E7, and recent evidence implicate also E5 are involved in viraltransformation.

In the HPVs involved in cervical carcinoma such as HPV 16 and 18, theoncogenic process starts after integration of viral DNA. The integrationresults in the inactivation of genes coding for the capsid proteins L1and L2 and in installing continuous over expression of the two earlyproteins E6 and E7 that will lead to gradual loss of the normal cellulardifferentiation and the development of the carcinoma.

Carcinoma of the cervix is common in women and develops through apre-cancerous intermediate stage to the invasive carcinoma whichfrequently leads to death. The intermediate stages of the disease isknown as cervical intraepithelial neoplasia and is graded I to III interms of increasing severity.

Clinically, HPV infection of the female anogenital tract manifests ascervical flat condylomas, the hallmark of which is the koilocytosisaffecting predominantly the superficial and intermediate cells of thecervical squamous epithelium.

Koilocytes which are the consequence of a cytopathic effect of thevirus, appear as multinucleated cells with a perinuclear clear halo. Theepithelium is thickened with abnormal keratinisation responsible for thewarty appearance of the lesion.

Such flat condylomas when positive for the HPV 16 or 18 serotypes, arehigh-risk factors for the evolution toward cervical intraepithelialneoplasia (CIN) and carcinoma in situ (CIS) which are themselvesregarded as precursor lesions of invasive cervix carcinoma.

WO 96/19496 discloses variants of human papillomavirus E6 and E7proteins, particularly fusion proteins of E6/E7 with a deletion in boththe E6 and E7 proteins. These deletion fusion proteins are said to beimmunogenic.

HPV L1 based vaccines are disclosed in WO94/00152, WO94/20137,WO93/02184 and WO94/05792. Such a vaccine can comprise the L1 antigen asa monomer, a capsomer or a virus like particle. Such particles mayadditionally comprise L2 proteins. L2 based vaccines are described forexample in WO93/00436. Other HPV vaccines are based on the Earlyproteins, such as E7 or fusion proteins such as L2-E7.

Vaccines for the prophylaxis of hepatitis B infections, comprising oneor more hepatitis B antigens, are well known. For example the vaccineEngerix-B (Trade Mark) from SmithKline Beecham Biologicals is used toprevent Hepatitis B. This vaccine comprises hepatitis B surface antigen(specifically the 226 amino acid S-antigen described in Harford et. al.in Postgraduate Medical Journal, 1987, 63 (Suppl. 2), p65-70) and isformulated using aluminum hydroxide as adjuvant.

There is a need for effective combination vaccines to prevent diseasesto which adolescents are particularly prone.

The present invention provides a vaccine composition comprising:

-   -   (a) a hepatitis B viral (HBV) antigen; and    -   (b) a human papillomavirus (HPV) antigen        in combination with an adjuvant which is a preferential        stimulator of TH1 cell response.

The vaccine composition of the invention is of great benefit foradministration to adolescents who may be particularly at risk of HBV,and/or HPV infection.

Optionally the vaccine composition of the invention additionallycomprises one or more of a number of other antigens as described below.

It has been found that the vaccine compositions according to theinvention surprisingly show no interference, that is to say that theimmune response to each antigen in the composition of the invention isessentially the same as that which is obtained by each antigen givenindividually in conjunction with an adjuvant which is a preferentialstimulator of TH1 cell response.

The vaccine Havrix (Trade Mark), also from SmithKline BeechamBiologicals is an example of a vaccine that can be used to preventhepatitis A infections. It is formulated with aluminium hydroxide asadjuvant. This vaccine comprises an attenuated strain of the HM-175Hepatitis A virus inactivated with formol (formaldehyde); see Andre et.al. (Prog. med. Virol., vol. 37, p1-24).

As used herein, the term hepatitis A viral (HAV) antigen is used torefer to either a protein derived from hepatitis A virus or anattenuated strain of HAV, optionally inactivated, e.g. withformaldehyde. If the HAV antigen is a protein derived from hepatitis Avirus it may optionally be a recombinant protein.

The vaccine Twinrix (Trade Mark) is a combination of a recombinanthepatitis B antigen with the aforementioned inactivated attenuatedhepatitis A virus. The vaccine may be used to protect against hepatitisA and hepatitis B simultaneously.

European patent 0 339 667 (Chemo Sero) describes the general concept ofcombining a hepatitis A antigen and a hepatitis B antigen to make acombination vaccine. In that specification it is stated that theadjuvant which is used is not critical: it must only be capable ofenhancing the immune activity to a desired extent and not cause anyside-effects. It is stated that aluminium gel may be used, in particularaluminium hydroxide gel and aluminium phosphate gel.

In a further aspect, the invention provides a vaccine compositioncomprising:

-   -   (a) a hepatitis B viral (HBV) antigen;    -   (b) a human papillomavirus (HPV) antigen; and    -   (c) a hepatitis A viral (HAV) antigen        in combination with an adjuvant which is a preferential        stimulator of TH1 cell response.

Such a vaccine is of great benefit for administration to adolescents whomay be particularly at risk of HBV, and/or HPV infection, and/or HAVinfection.

An immune response may be broadly divided into two extreme catagories,being a humoral or cell mediated immune response (traditionallycharacterized by antibody and cellular effector mechanisms of protectionrespectively). These categories of response have been termed TH1-typeresponses (cell-mediated response), and TH2-type immune responses(humoral response).

Extreme TH1-type immune responses may be characterized by the generationof antigen specific, haplotype restricted cytotoxic T lymphocytes, andnatural killer cell responses. In mice TH1-type responses are oftencharacterized by the generation of antibodies of the IgG2a subtype,whilst in the human these correspond to IgG1 type antibodies. TH2-typeimmune responses are characterized by the generation of a range ofimmunoglobulin isotypes including in mice IgG1.

It can be considered that the driving force behind the development ofthese two types of immune responses are cytokines. High levels ofTH1-type cytokines tend to favour the induction of cell mediated immuneresponses to the given antigen, whilst high levels of TH2-type cytokinestend to favour the induction of humoral immune responses to the antigen.

The distinction of TH1 and TH2-type immune responses is not absolute. Inreality an individual will support an immune response which is describedas being predominantly TH1 or predominantly TH2. However, it is oftenconvenient to consider the families of cytokines in terms of thatdescribed in murine CD4+ve T cell clones by Mosmann and Coffman(Mosmann, T. R. and Coffman, R. L. (1989) TH1 and TH2 cells: differentpatterns of lymphokine secretion lead to different functionalproperties. Annual Review of Immunology, 7, p145-173). Traditionally,TH1-type responses are associated with the production of the INF-γcytokines by T-lymphocytes. Other cytokines often directly associatedwith the induction of TH1-type immune responses are not produced byT-cells, such as IL-12. In contrast, TH2-type responses are associatedwith the secretion of IL4, IL-5, IL-6, IL-10 and tumour necrosisfactor-β(TNF-β).

It is known that certain vaccine adjuvants are particularly suited tothe stimulation of either TH1 or TH2-type cytokine responses.Traditionally the best indicators of the TH1:TH2 balance of the immuneresponse after a vaccination or infection includes direct measurement ofthe production of TH1 or TH2 cytokines by T lymphocytes in vitro afterrestimulation with antigen, and/or the measurement (at least in mice) ofthe IgG1:IgG2a ratio of antigen specific antibody responses.

Thus, a TH1-type adjuvant is one which stimulates isolated T-cellpopulations to produce high levels of TH1-type cytokines whenre-stimulated with antigen in vitro, and induces antigen specificimmunoglobulin responses associated with TH1-type isotype.

Adjuvants which are capable of preferential stimulation of the TH1 cellresponse are described in International Patent Application No. WO94/00153 and WO 95/17209. 3 De-O-acylated monophosphoryl lipid A(3D-MPL) is one such adjuvant. This is known from GB 2220211 (Ribi).Chemically it is a mixture of 3 De-O-acylated monophosphoryl lipid Awith 4, 5 or 6 acylated chains and is manufactured by Ribi Immnunochem,Montana. A preferred form of 3 De-O-acylated monophosphoryl lipid A isdisclosed in European Patent 0 689 454 B1 (SmithKline BeechamBiologicals SA).

Preferably, the particles of 3D-MPL are small enough to be sterilefiltered through a 0.22 micron membrane (as described in European Patentnumber 0 689 454). 3D-MPL will be present in the range of 10 μg-100 μgpreferably 25-50 μg per dose wherein the antigen will typically bepresent in a range 2-50 μg per dose.

Another preferred adjuvant comprises QS21, an Hplc purified non-toxicfraction derived from the bark of Quillaja Saponaria Molina. Optionallythis may be admixed with 3 De-O-acylated monophosphoryl lipid A(3D-MPL), optionally together with an carrier.

The method of production of QS21 is disclosed in U.S. Pat. No.5,057,540.

Non-reactogenic adjuvant formulations containing QS21 have beendescribed previously (WO 96/33739). Such formulations comprising QS21and cholesterol have been shown to be successful TH1 stimulatingadjuvants when formulated together with an antigen. Thus vaccinecompositions which form part of the present invention may include acombination of QS21 and cholesterol.

Further adjuvants which are preferential stimulators of TH1 cellresponse include immunomodulatory oligonucleotides, for exampleunmethylated CpG sequences as disclosed in WO 96/02555.

Combinations of different TH1 stimulating adjuvants, such as thosementioned hereinabove, are also contemplated as providing an adjuvantwhich is a preferential stimulator of TH1 cell response. For example,QS21 can be formulated together with 3D-MPL. The ratio of QS21:3D-MPLwill typically be in the order of 1:10 to 10:1; preferably 1:5 to 5:1and often substantially 1:1. The preferred range for optimal synergy is2.5:1 to 1:1 3D-MPL: QS21.

Preferably a carrier is also present in the vaccine compositionaccording to the invention. The carrier may be an oil in water emulsion,or an aluminium salt, such as aluminium phosphate or aluminiumhydroxide.

A preferred oil-in-water emulsion comprises a metabolisible oil, such assqualene, alpha tocopherol and Tween 80. Additionally the oil in wateremulsion may contain span 85 and/or lecithin and/or tricaprylin.

In a particularly preferred aspect the antigens in the vaccinecomposition according to the invention are combined with 3D-MPL andalum.

Typically for human administration QS21 and 3D-MPL will be present in avaccine in the range of 1 μg-200 μg, such as 10-100 μg, preferably 10μg-50 μg per dose. Typically the oil in water will comprise from 2 to10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween80. Preferably the ratio of squalene: alpha tocopherol is equal to orless than 1 as this provides a more stable emulsion. Span 85 may also bepresent at a level of 1%. In some cases it may be advantageous that thevaccines of the present invention will further contain a stabiliser.

Non-toxic oil in water emulsions preferably contain a non-toxic oil,e.g. squalane or squalene, an emulsifier, e.g. Tween 80, in an aqueouscarrier. The aqueous carrier may be, for example, phosphate bufferedsaline.

A particularly potent adjuvant formulation involving QS21, 3D-MPL andtocopherol in an oil in water emulsion is described in WO 95/17210.

The HPV antigen in the composition of the invention is preferablyderived from HPV 16 and/or 18, or from HPV 6 and/or 11, or HPV 31, 33 or45.

In one preferred embodiment the HPV antigen in the vaccine compositionaccording to the invention comprises the major capsid protein L1 of HPVand optionally the L2 protein, particularly from HPV 16 and/or HPV 18.In this embodiment, the preferred form of the L1 protein is a truncatedL1 protein. Preferably the L1 is in the form of a virus-like particle(VLP). The L1 protein may be fused to another HPV protein, in particularE7 to form an L1-E7 fusion. Chimeric VLPs comprising L1-E or L1-L2-E areparticularly preferred.

In another preferred embodiment, the HPV antigen in the composition ofthe invention is derived from an E6 or E7 protein, in particular E6 orE7 linked to an immunological fusion partner having T cell epitopes.

In a preferred form of this embodiment of the invention, theimmunological fusion partner is derived from protein D of Heamophilusinfluenza B. Preferably the protein D derivative comprises approximatelythe first 1/3 of the protein, in particular approximately the firstN-terminal 100-110 amino acids.

Preferred fusion proteins in this embodiment of the invention compriseProtein D-E6 from HPV 16, Protein D-E7 from HPV 16 Protein D-E7 from HPV18 and Protein D-E6 from HPV 18. The protein D part preferably comprisesthe first 1/3 of protein D.

In still another embodiment of the invention, the HPV antigen is in theform of an L2-E7 fusion, particularly from HPV 6 and/or HPV 11.

The proteins of the present invention preferably are expressed in E.coli. In a preferred embodiment the proteins are expressed with aHistidine tail comprising between 5 to 9 and preferably six Histidineresidues. These are advantageous in aiding purification. The descriptionof the manufacture of such proteins is fully described in co-pending UKpatent application number GB 9717953.5.

The HPV antigen in the vaccine composition may be adsorbed onto Al(OH)₃.Preferably the L1 VLP is adsorbed onto Al(OH)₃.

The hepatitis B viral (HBV) antigen in the composition of the inventionis typically hepatitis B surface antigen.

The preparation of Hepatitis B surface antigen (HBsAg) is welldocumented. See for example, Harford et. al. in Develop. Biol. Standard54, page 125 (1983), Gregg et. al. in Biotechnology, 5, page 479 (1987),EP-A-0 226 846, EP-A-0 299 108 and references therein.

As used herein the expression ‘Hepatitis B surface antigen’, abbreviatedherein to ‘HBsAg’ or ‘HBS’ includes any HBsAg antigen or fragmentthereof displaying the antigenicity of HBV surface antigen. It will beunderstood that in addition to the 226 amino acid sequence of the HBsAgS antigen (see Tiollais et. al. Nature, 317, 489 (1985) and referencestherein) HBsAg as herein described may, if desired, contain all or partof a pre-S sequence as described in the above references and in EP-A-0278 940. HBsAg as herein described can also refer to variants, forexample the ‘escape mutant’ described in WO 91/14703. In a furtheraspect the HBsAg may comprise a protein described as L* in EuropeanPatent Application Number 0 414 374, that is to say a protein, the aminoacid sequence of which consists of parts of the amino acid sequence ofthe hepatitis B virus large (L) protein (ad or ay subtype),characterized in that the amino acid sequence of the protein consists ofeither:

-   -   (a) residues 12-52, followed by residues 133-145, followed by        residues 175-400 of the said L protein; or    -   (b) residue 12, followed by residues 14-52, followed by residues        133-145, followed by residues 175-400 of the said L protein.

HBsAg may also refer to polypeptides described in EP 0 198 474 or EP 0304 578.

Normally the HBsAg will be in particle form. It may comprise S proteinalone or may be as composite particles, for example (L*,S) wherein L* isas defined above and S denotes the S-protein of hepatitis B surfaceantigen.

The HBsAg may be adsorbed on aluminium phosphate as described inWO93/24148.

Preferably the hepatitis B (HBV) antigen used in the formulation of theinvention is HBsAg S-antigen as used in the commercial product Engerix-B(Trade Mark; SmithKline Beecham Biologicals).

A vaccine comprising hepatitis B surface antigen in conjunction with3D-MPL was described in European Patent Application 0 633 784.

Examples of antigens from additional pathogens which may be included inthe compositions according to the invention are now described.

Epstein Barr Virus (EBV), a member of the herpes virus group, causesinfectious mononucleosis as a primary disease in humans. Predominantlyit affects children or young adults. More than 90% of the average adultpopulation is infected by EBV that persists for lifetime in peripheralB-lymphocytes. The virus is lifelong produced in the parotid gland andspread primarily by exchange of saliva from individuals who shed thevirus. Children infected with EBV are largely asymptomatic or have verymild symptoms, while adolescents and adults who become infected developtypical infectious mononucleosis, characterized by fever, pharyngitis,and adenopathy. People who have been infected maintain anti-EBVantibodies for the remainder of their lives, and are thus immune tofurther infection.

In addition to its infectious qualities, EBV has been shown to transformlymphocytes into rapidly dividing cells and has therefore beenimplicated in several different lymphomas, including African Burkitt'slymphoma (BL). EBV may also be involved in causing nasopharyngealcarcinoma (NPC). Worldwide it is estimated that 80,000 cases ofnasopharyngeal carcinoma occur and it is more prevalent in ethnicChinese populations. Infectious mononucleosis is a consequence ofprimary infection by EBV. It is not a life-threatening disease ifadditional risk factors are absent.

Four proteins of the EBV viral envelope constituting the so-calledmembrane antigen complex have been described. They are usually referredto as gp 220/350 or gp 250/350 or simply as gp 250 or 350 (seeEP-A-151079). There is convincing evidence that gp 350 and gp 250 inducethe production of neutralising antibodies and that antibodies against gp350 and gp 250 have neutralising capacity. These proteins are thuscandidates for a possible EBV vaccine. For further information about theapplication of gp 250/350 for prophylaxis and treatment of EBV-relateddiseases see EP 0 173 254.

The major EBV surface glycoprotein gp350/220 infects human target cellsthrough interaction with the cellular membrane protein, CD21. Gp350/220is the primary target for EBV-neutralising antibodies in humans and someforms of gp350/220 have been shown to protect against EBV-relateddisease. Preferably a vaccine composition according to the inventioncomprises gp 350 of EBV although other protective antigens may be used.

HSV-2 is the primary etiological agent of herpes genitalis. HSV-2 andHSV-1 (the causative agent of herpes labialis) are characterized bytheir ability to induce both acute diseases and to establish a latentinfection, primarily in neuronal ganglia cells.

Genital herpes is estimated to occur in about 5 million people in theU.S.A. alone with 500,000 clinical cases recorded every year (primaryand recurrent infection). Primary infection typically occurs afterpuberty and is characterized by the localised appearance of painful skinlesions, which persist for a period of between 2 to 3 weeks. Within thefollowing six months after primary infection 50% of patients willexperience a recurrence of the disease. About 25% of patients mayexperience between 10-15 recurrent episodes of the disease each year. Inimmunocompromised patients the incidence of high frequency recurrence isstatistically higher than in the normal patient population.

Both HSV-1 and HSV-2 virus have a number of glycoprotein componentslocated on the surface of the virus. These are known as gB, gC, gD andgE etc.

When an HSV antigen is included in the composition of the invention thisis preferably derived from HSV-2, typically glycoprotein D. GlycoproteinD is located on the viral membrane, and is also found in the cytoplasmof infected cells (Eisenberg R. J. et al; J of Virol 1980, 35, 428-435).It comprises 393 amino acids including a signal peptide and has amolecular weight of approximately 60 kD. Of all the HSV envelopeglycoproteins this is probably the best characterized (Cohen et al; J.of Virology, 60, 157-166). In vivo it is known to play a central role inviral attachment to cell membranes. Moreover, glycoprotein D has beenshown to be able to elicit neutralising antibodies in vivo (Eing et alJ. Med. Virology 127: 59-65). However, latent HSV-2 virus can still bereactivated and induce recurrence of the disease despite the presence ofhigh neutralising antibodies titre in the patients sera.

In one embodiment of the invention there is present a truncated HSV-2glycoprotein D of 308 amino acids which comprises amino acids 1 through306 naturally occurring glycoprotein with the addition Asparagine andGlutamine at the C terminal end of the truncated protein devoid of itsmembrane anchor region. This form of the protein includes the signalpeptide which is cleaved to yield a mature 283 amino acid protein. Theproduction of such a protein in Chinese Hamster ovary cells has beendescribed in Genentech's European patent EP-B-139 417.

The recombinant mature HSV-2 glycoprotein D truncate is preferably usedin the vaccine formulations of the present invention and is designatedrgD2t.

A combination of this HSV-2 antigen in combination with the adjuvant3D-MPL has been described in WO 92/16231.

In a preferred aspect the vaccine composition of the inventionadditionally comprises a Varicella Zoster viral antigen (VZV antigen).Suitable antigens of VZV for inclusion in the vaccine formulationinclude gpI-V described by Longnecker et al., Proc Natl Acad Sci USA 84,4303-4307 (1987).

In a preferred embodiment gpI (see Ellis et al., U.S. Pat. No.4,769,239) is used. See also European Patent No. 0 405 867 B1.

In another preferred aspect the vaccine composition of the inventionadditionally comprises a human cytomegalovirus (HCMV) antigen. HCMV is ahuman DNA virus belonging to the family of herpes viruses. HCMV isendemic in most parts of the world. Among two populations, HCMV isresponsible for serious medical conditions. HCMV is a major cause ofcongenital defects in new borns. The second population at risk areimmunocompromised patients such as those suffering from HIV infectionand those patients undergoing transplantations. The clinical diseasecauses a variety of symptoms including fever, hepatitis, pneumonitis andinfectious mononucleosis. A preferred antigen for use in a vaccineagainst HCMV is gB685** as described in WO 95/31555. Immunogens for usein HCMV vaccines are also provided by pp65, an HCMV Matrix Protein asdescribed in WO 94/00150 (City of Hope).

In one preferred aspect the vaccine composition of the inventionadditionally comprises both a VZV and an HCMV antigen, in particularthose antigens described above.

In another preferred aspect the vaccine composition of the inventionadditionally comprises a Toxoplasma gondii antigen. Toxoplasma gondii isan obligate intracellular protozoan parasite responsible fortoxoplasmosis in warm-blooded animals, including man. Although it isgenerally clinically asymptomatic in healthy individuals, toxoplasmosismay cause severe complications in pregnant women and immunocompromisedpatients. A preferred antigen for use in a vaccine against Toxoplasmagondii is SAG1 (also known as P30) as described in WO96/02654 or Tg34 asdescribed in WO92/11366.

In one preferred aspect the vaccine composition of the inventionadditionally comprises either a VZV antigen or an HCMV antigen combinedwith a Toxoplasma gondii antigen, in particular those antigens describedabove.

In a preferred aspect the vaccine composition of the invention is amultivalent vaccine, for example a tetra-or pentavalent vaccine.

The formulations of the present invention are very effective in inducingprotective immunity, even with very low doses of antigen (e.g. as low as5 μg rgD2t).

They provide excellent protection against primary infection andstimulate, advantageously both specific humoral (neutralisingantibodies) and also effector cell mediated (DTH) immune responses.

The present invention in a further aspect provides a vaccine formulationas herein described for use in medical therapy, particularly for use inthe treatment or prophylaxis of human papillomavirus infections andhepatitis B virus infections.

The vaccine of the present invention will contain an immunoprotectivequantity of the antigens and may be prepared by conventional techniques.

Vaccine preparation is generally described in PharmaceuticalBiotechnology, Vol.61 Vaccine Design—the subunit and adjuvant approach,edited by Powell and Newman, Plenurn Press, 1995. New Trends andDevelopments in Vaccines, edited by Voller et al., University ParkPress, Baltimore, Md., U.S.A. 1978. Encapsulation within liposomes isdescribed, for example, by Fullerton, U.S. Pat. No. 4,235,877.Conjugation of proteins to macromolecules is disclosed, for example, byLikhite, U.S. Pat. No. 4,372,945 and by Armor et al., U.S. Pat. No.4,474,757.

The amount of protein in each vaccine dose is selected as an amountwhich induces an immunoprotective response without significant, adverseside effects in typical vaccinees. Such amount will vary depending uponwhich specific immunogen is employed. Generally, it is expected thateach dose will comprise 1-1000 μg of protein, preferably 2-100 μg, mostpreferably 4-40 μg. An optimal amount for a particular vaccine can beascertained by standard studies involving observation of antibody titresand other responses in subjects. Following an initial vaccination,subjects may receive a boost in about 4 weeks.

In addition to vaccination of persons susceptible to HPV or HBVinfections, the pharmaceutical compositions of the present invention maybe used to treat, immunotherapeutically, patients suffering from thesaid viral infections.

In a further aspect of the present invention there is provided a methodof manufacture as herein described, wherein the method comprises mixinga human papilloma virus antigen and a hepatitis B virus antigen with aTH-1 inducing adjuvant, for example 3D-MPL and, preferably, a carrier,for example alum.

If desired, other antigens may be added, in any convenient order, toprovide multivalent vaccine compositions as described herein.

The following example illustrates but does not limit the invention.

EXAMPLE 1 Comparative Immunogenicity of HPV Ags/HBs Combos Fomulatedwith Alum/3D-MPL

Introduction

An immunogenicity study was performed in Balb/C mice using fourdifferent antigens:

-   -   1. HPV16 L1 Virus Like Particule (VLP-16)    -   2. HPV18 L1 Virus Like Particule (VLP18)    -   3. PD 1/3 16E7 2M from HPV-16 (E7)    -   4. HBsAg        formulated with Alum/3D-MPL (AS04) using pre adsorbed monobulks        of antigen or 3D-MPL on Al(OH)₃ or AlPO₄.

3D-MPL/Al(OH)₃ formulations are referred to as AS04D whereas3D-MPL/AlPO₄ based formulations are referred to as AS04C.

The following vaccines were assessed:

-   -   1. VLP16+VLP18 AS04D;    -   2. E7 based formulations,    -   3. HBs AS04C        and the potential to combine these vaccines was evaluated.

The aims of this experiment were as follows:

-   -   1) To compare the immunogenicity of different AS04 combinations        of either VLP16+VLP18 or E7 and HBs Ag.    -   2) As the monovalent vaccines are either formulated in AS04C or        AS04D:        -   to compare the immunogenicity of different HBs AS04            formulations made of AlPO₄ or a mix of AlPO₄/Al(OH)₃ with            different ratios of Alum forms; and        -   to evaluate the effect of the adsorption of 3D-MPL on a            fraction of Al(OH)₃ and AlPO₄ versus 3D-MPL/Al(OH)₃ in            combination containing VLPs or E7 antigens.

The experimental protocol is fully described in the Material and Methodssection.

In summary, groups of 10 mice were immunised intramuscularly twice aweek at 3 week intervals with various Ag based formulations (1/10HD).Antibody response to HBs, E7 and VLPs Ag and the isotypic profileinduced by vaccination were monitored by ELISA at day 14 post II. At thesame timepoint, the CMI (lymphoproliferative response or the cytokineproduction (IFNγ/IL5)) was analysed after in vitro restimulation ofsplenic cells with either HBs, VLPs or E7 antigen.

Materials and Methods

Formulation

Formulation Compositions

VLP16, VLP18, PD1/3-HPV16E7-His, and HBs on AS04C or AS04D.

Components used Component Concentration Buffer HPV 16 VLP  560 μg/mlTris 20 mM/NaCl 500 mM HPV 18 VLP  550 μg/ml NaCl 500 mM/NaPO₄ 20 mMAL(OH)₃ 10380 μg/ml H₂O PD1/3-HPV 16 E7-  1170 μg/ml PO₄ 20 mM His HBs 1219 μg/ml PO₄ 10 mM/NaCl 150 mM 3D-MPL  1170 μg/ml Water For InjectionAlPO₄   5 mg/ml NaCl 150 mMAdsorptiona) VLP Adsorption.

VLP 16 and VLP 18 purified bulk is added to Al(OH)₃ at 2 μg VLP/10 μgAl(OH)₃. The mixture is stored between 2-8° C. until final formulation.

b) HBs Adsorption.

2 μg Hbs are mixed with 40 μg AlPO₄. The mixture is stored between 2-8°C. until final formulation.

2 μg Hbs are mixed with 10 μg AlPO₄. The mixture is stored between 2-8°C. until final formulation.

c) PD1/3-HPV16E7-His Adsorption.

2 μg E7 are mixed with 10 μg Al(OH)₃. The mixture is stored between 2-8°C. until final formulation.

d) 3D-MPL Adsorption.

5 μg 3D-MPL are mixed with 10 μg Al(OH)₃. The mixture is stored between2-8° C. until final formulation.

5 μg 3D-MPL are mixed with 10 μg AlPO₄. The mixture is stored between2-8° C. until final formulation.

2.5 μg 3D-MPL are mixed with 5 μg Al(OH)₃. The mixture is stored between2-8° C. until final formulation.

2.5 μg 3D-MPL are mixed with 5 μg AlPO₄. The mixture is stored between2-8° C. until final formulation.

Formulation

H₂O and NaCl are mixed (10× concentrated) and after 10 minutes ofagitation at room temperature, the different components are added:adsorbed antigen, adsorbed 3D-MPL and Al(OH)₃ (See table below). Theyare shaken at room temperature for 10 minutes and stored at 4° C. untilinjection. The in vitro characterization of the formulation can then beperformed.

Table of groups and details of formulations Antigen(s) ImmunostimulantsVehicle Group Type μg Type μg Type μg A VLP16 2 Al(OH)₃ 10 VLP18 2Al(OH)₃ 10 3D-MPL 5 Al(OH)₃ 10 20 B HPV16E7 2 Al(OH)₃ 10 3D-MPL 5Al(OH)₃ 10 30 C HBs 2 AlPO₄ 40 3D-MPL 5 AlPO₄ 10 D HBs 2 AlPO₄ 10 3D-MPL5 AlPO₄ 10 Al(OH)₃ 30 E HBs 2 AlPO₄ 10 3D-MPL 5 Al(OH)₃ 10 Al(OH)₃ 30 FE7 2 Al(OH)₃ 10 HBs 2 AlPO₄ 10 3D-MPL 5 Al(OH)₃ 10 Al(OH)₃ 20 G VLP16 2Al(OH)₃ 10 VLP18 2 Al(OH)₃ 10 HBs 2 AlPO₄ 10 3D-MPL 5 Al(OH)₃ 10 Al(OH)₃10 H VLP16 2 Al(OH)₃ 10 VLP18 2 Al(OH)₃ 10 HBs 2 AlPO₄ 10 3D-MPL 2.5Al(OH)₃ 5 3D-MPL 2.5 AlPO₄ 5 Al(OH)₃ 10Mice SerologyAnti-HBs Serology

The quantitation of anti-HBs antibodies was performed by ELISA using HBs(Hep 286) as the coating antigen. Antigen and antibody solutions wereused at 50 μl per well. The antigen was diluted at a final concentrationof 1 μg/ml in PBS and was adsorbed overnight at 4° C. to the wells of 96wells microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark). Theplates were then incubated for 1 hr at 37° C. with PBS containing 1%bovine serum albumin and 0.1% Tween 20 (saturation buffer). Two-folddilutions of sera (starting at 1/100 dilution) in the saturation bufferwere added to the HBs-coated plates and incubated for 1 hr 30 min at 37°C. The plates were washed four times with PBS 0.1% Tween 20 andbiotin-conjugated anti-mouse Ig (Amersham, UK) diluted 1/1500 or IgG1,IgG2a, IgG2b (IMTECH, USA) diluted respectively at 1/4000, 1/8000,1/4000 in saturation buffer were added to each well and incubated for 1hr 30 min at 37° C. After a washing step, streptavidin-biotinylatedperoxydase complex (Amersham, UK) diluted 1/1000 in saturation bufferwas added for an additional 30 min at 37° C. Plates were washed as aboveand incubated for 20 min with a solution of o-phenylenediarnine (Sigma)0.04% H₂O₂ 0.03% in 0.1% tween 20 0.05M citrate buffer pH4.5. Thereaction was stopped with H₂SO₄ 2N and read at 490/630 nm. ELISA titerswere calculated from a reference by SoftmaxPro (using a four parametersequation) and expressed in EU/ml.

Anti-E7 Serology

Quantitation of anti-E7 antibody was performed by ELISA using PD1/3 16E72M as coating antigen. Antigen and antibody solutions were used at 100μl per well. The antigen was diluted at a final concentration of 0.5μg/ml in PBS and was adsorbed overnight at 4° C. to the wells of 96wells microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark). Theplates were then incubated for 1 hr at 37° C. with PBS containing 1%bovine serum albumin and 0.1% Tween 20 (saturation buffer). Two-folddilutions of sera (starting at 1/100 or 1/400 dilution) in thesaturation buffer were added to the E7-coated plates and incubated for 1hr 30 min at 37° C. The plates were washed four times with PBS 0.1%Tween 20 and biotin-conjugated anti-mouse Ig, IgG1, IgG2a, IgG2b(Amersham, UK) diluted 1/1500 in saturation buffer were added to eachwell and incubated for 1 hr 30 min at 37° C. After washing step,streptavidin-biotinylated peroxydase complex (Amersham, UK) diluted1/5000 in saturation buffer was added for an additional 30 min at 37° C.Plates were washed as above and incubated for 20 min with a solution ofTetramethyl benzidine (TMB) (Biorad, USA) 2-fold diluted in Citratebuffer (0.1M pH=5.8). The reaction was stopped with H₂SO₄ 0.5 N and readat 450/630 nm. ELISA titers were calculated from a reference bySoftmaxPro (using a four parameters equation) and expressed in EU/ml.

Anti-VLP16 and Anti-VLP18 Serology

The quantitation of anti-VLP16 and anti-VLP18 antibodies was performedby ELISA using VLP16 503/1 (20/12/99) ands VLP18 504/2 (25/10/99F) ascoating antigens. The antigen and antibody solutions were used at 50 μlper well. The antigen was diluted at a final concentration of 0.5 μg/mlin PBS and was adsorbed overnight at 4° C. to the wells of 96 wellsmicrotiter plates (Maxisorb Immuno-plate, Nunc, Denmark). The plateswere then incubated for 1 hr at 37° C. with PBS containing 1% bovineserum albumin. Two-fold dilutions of sera (starting at 1/400 dilution)in the saturation buffer were added to the VLPs-coated plates andincubated for 1 hr 30 min at 37° C. The plates were washed four timeswith PBS 0.1% Tween 20 and biotin-conjugated anti-mouse Ig (Amersham,UK) diluted 1/1500 in saturation buffer were added to each well andincubated for 1 hr 30 min at 37° C. After a washing step,streptavidin-biotinylated peroxydase complex (Amersham, UK) diluted1/1000 in saturation buffer was added for an additional 30 min at 37° C.Plates were washed as above and incubated for 20 min with a solution ofo-phenylenediamine (Sigma) 0.04% H₂O₂ 0.03% in 0.1% tween 20 0.05Mcitrate buffer pH4.5. The reaction was stopped with H₂SO₄ 2N and read at490/630 nm. ELISA titers were calculated from a reference by SoftmaxPro(using a four parameters equation) and expressed in EU/ml.

T Cell Proliferation

Two weeks after the second immunisation, mice were killed, spleens wereremoved aseptically and pooled (1 pool of 5 organs per group). Cellsuspensions were prepared in RPMI 1640 medium (GIBCO) containing 2 mML-glutamine, antibiotics, 5×10⁻⁵M 2-mercaptoethanol, and 1% syngeneicnormal mouse serum. Splenic cells were cultured at a final concentrationof 2×10⁶ cells/ml in 200 μl in round-bottomed 96 wells-plates withdifferent concentrations (10-0.03 μg/ml) of each of the Ag (VLPs, E7 orHBs antigen). Each test was carried out in quadriplicate. After 96 hr ofculture at 37° C. under 5% CO₂, the cells were pulsed for 18 hr with³H-Thymidine (Amersham, UK, 5 Ci/mmol) at 0.5 μCi/well and thenharvested on Unifilter plates (Packard) with a cell harvester.Incorporated radioactivity was measured in a scintillation counter(Topcount, Packard). Results are expressed in cpm (mean cpm inquadriplicate wells) or as stimulation indices (mean cpm in cultures ofcells with antigen/mean cpm in cultures of cells without antigen).

Cytokine Production

Two weeks after the second immunisation, mice were killed, spleens wereremoved aseptically and pooled. Cell suspensions were prepared in RPMI1640 medium (GIBCO) containing 2 mM L-glutamine, antibiotics, 5×10⁻⁵M2-mercaptoethanol, and 5% foetal calf serum. Cells were cultured at afinal concentration of 5×10⁶ cells/ml, in 1 ml per flat-bottomed 24wells-plates with different concentrations (10-1 μg/ml) of each of theAg (VLPs, E7 or HBs antigen). Supernatants were harvested 96 hrs laterand frozen until tested for the presence of IFNγ and IL5 by Elisa.

IFNγ (Genzyme)

Quantitation of IFNγ was performed by Elisa using reagents from Genzyme.Samples and antibody solutions were used at 50 μl per well. 96-wellsmicrotiter plates (Maxisorb Immuno-plate, Nunc, Denmark) were coatedovernight at 4° C. with 50 μl of hamster anti-mouse IFNγ diluted at 1.5μg/ml in carbonate buffer pH 9.5. Plates were then incubated for 1 hr at37° C. with 100 μl of PBS containing 1% bovine serum albumin and 0.1%Tween 20 (saturation buffer). Two-fold dilutions of supernatant from invitro stimulation (starting at 1/2) in saturation buffer were added tothe anti-IFNγ-coated plates and incubated for 1 hr 30 min at 37° C. Theplates were washed 4 times with PBS Tween 0.1% (wash buffer) andbiotin-conjugated goat anti-mouse IFNγ diluted in saturation buffer at afinal concentration of 0.5 μg/ml was added to each well and incubatedfor 1 hr at 37° C. After a washing step, AMDEX conjugate (Amersham)diluted 1/10000 in saturation buffer was added for 30 min at 37° C.Plates were washed as above and incubated with 50 μl of TMB (Biorad) for10 min. The reaction was stopped with H₂SO₄ 0.4N and read at 450/630 nm.Concentrations were calculated using a standard curve (mouse IFNγstandard) by SoftmaxPro (four parameters equation) and expressed inpg/ml.

IL5 (Pharmingen)

Quantitation of IL5 was performed by Elisa using reagents fromPharmingen. Samples and antibody solutions were used at 50 μl per well.96-wells microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark) werecoated overnight at 4° C. with 50 μl of rat anti-mouse IL5 diluted at 1μg/ml in carbonate buffer pH 9.5. Plates were then incubated for 1 hr at37° C. with 100 μl PBS containing 1% bovine serum albumin and 0.1% tween20 (saturation buffer). Two-fold dilutions of supernatant from in vitrostimulation (starting at 1/2) in saturation buffer were added to theanti-IL-5-coated plates and incubated for 1 hr 30 min at 37° C. Theplates were washed 4 times with PBS Tween 0.1% (wash buffer) andbiotin-conjugated rat anti-mouse IL5 diluted in saturation buffer at afinal concentration of 1 μg/ml was added to each well and incubated for1 hr at 37° C. After a washing step, AMDEX conjugate (Amersham) diluted1/10000 in saturation buffer was added for 30 min at 37° C. Plates werewashed as above and incubated with 50 μl of TMB (Biorad) for 15 min. Thereaction was stopped with H₂SO₄ 0.4N and read at 450/630 nm.Concentrations were calculated using a standard curve (recombinant mouseIL-5) by SoftmaxPro (four parameters equation) and expressed in pg/ml.

Groups

Groups of 10 Balb/C mice were immunised intramuscularly with thefollowing formulations:

TABLE 1 Groups and formulations GROUP FORMULATION A VLP16 2 μg/VLP18 2μg/3D-MPL 5 μg/Al(OH)3 50 μg B 16E7 2 μg/3D-MPL 5 μg/Al(OH)3 50 μg C HBs2 μg/3D-MPL 5 μg/AlPO4 50 μg D HBs 2 μg/3D-MPL 5 μg/AlPO4 20 μg/Al(OH)330 μg E HBs 2 μg/3D-MPL 5 μg/AlPO4 10 μg/Al(OH)3 40 μg F 16E7 2 μg/HBS 2μg/3D-MPL 5 μg/Al(OH) 40 μg/AlPO4 10 μg G VLP16 2 μg/VLP 18 2 μg/HBs 2μg/3D-MPL 5 μg/Al(OH)3 40 μg/AlPO4 10 μg H VLP16 2 μg/VLP 18 2 μg/HBs 2μg/3D-MPL 5 μg/Al(OH)3 35 μg/AlPO4 15 μg

Details of formulation are described above in the table in Materials andMethods.

Results

1. Serology

a) Anti-HBs Response:

Humoral responses (Ig and isotypes) were measured by Elisa using HBsAg(Hep286) as coating antigen. Day 14 post II sera were analysed.

FIG. 1 shows the anti-HBs antibody responses measured on individual seraon day 14 post II.

No difference was observed in the anti-HBs antibody response between theprotocols applied to adsorb the 3D-MPL: on Al(OH)₃ alone or AlPO₄ alone(groups C, D, E) with different ratios of Al(OH)₃ and AlPO₄ in thevaccine (GMT of 27905 EU/ml versus 30832 or 26670 EU/ml).

A slightly lower anti-HBs antibody response is observed in thecombination groups G and H containing the VLPs and the HBs antigencompared to HBs alone (group C) (GMT respectively of 10635 or 15589EU/ml versus 27905 EU/ml). Anti-HBs GMT obtained in the E7/HBscombination reached 19235 EU/ml.

Before statistical analysis, a T-Grubbs test was applied on eachpopulation for data exclusion. One mouse in group C was eliminated foranalysis.

A one-way-analysis of variance was performed on anti-HBs titers afterlog transformation of post II data. Significant differences wereobserved between formulations (p-value=0.0108) and the Student NewmanKeuls test was then applied for multiple comparisons. No statisticallysignificant difference was observed between the group H (VLP/HBs) orgroup F (HBs/E7) combination versus the group C (HBs AS04C). Astatistically significant difference was shown between the group G(VLP/HBs) and the group C (HBs AS04C) (p value=0.0291) however the 95%confidence intervals of the 2 groups overlap and the difference whichreaches a 2.5 ratio might not be biologically relevant.

The isotypic repartition analysed on pooled sera was as follows andshowed no major differences between the 6 groups.

Isotypic repartition (%) IgG1 IgG2a IgG2b Group C 59 31 10 Group D 69 1912 Group E 66 19 15 Group F 61 22 17 Group G 61 30 9 Group H 46 29 25b) Anti-E7 Response:

Humoral responses (Ig and isotypes) were measured by Elisa using PD1/316E7 2M as the coating antigen. Day 14 post II sera of group B and Fwere analysed.

FIG. 2 shows the anti-E7 antibody responses measured on individual seraat day 14 post II:

A slight decrease was observed in the anti-E7 response with a two folddecrease in GMT for HBs/E7 combinations compared to E7 alone (9626versus 22447 EU/ml). This was established as statistically insignificantusing the Student Newman Keuls test.

No difference was observed in the isotypic profile induced by the twoformulations: mainly IgG1 response (97-98% of IgG1) as reported in thetable below.

The isotypic repartition analysed on pooled sera was as follows:

Isotypic repartition (%) IgG1 IgG2a IgG2b Group B 98 0 1 Group F 97 1 2c) Anti-VLP16 Response:

Humoral responses (Ig) were measured by Elisa using VLP16 503-1(20/12/99) as the coating antigen. Day 14 post II sera were analysed.

FIG. 3 shows anti-VLP16 Ig antibody responses measured on individualsera on day 14 post II.

Similar anti-VLP16 titers were obtained after immunisation with thecombination of HBs and VLPs (group G and H) as with the monovalent VLPsformulation (group A) (GMT of 19570 or 23448 EU/ml versus 30311 EU/ml)

Equivalent titers were observed between the two combinations preparedusing either ways to adsorb the 3D-MPL: Al(OH)₃ alone (group G) comparedto mixed adsorption on Al(OH)₃ and AlPO₄ (group H) (GMT of 19570 EU/mlversus 23448 EU/ml).

These differences were shown as statistically not significant usingone-way analysis of variance test.

d) Anti-VLP18 Response:

Humoral responses (Ig) were measured by Elisa using VLP18 504-2(25/10/99) as the coating antigen. Day 14 post II sera were analysed.

FIG. 4 shows the anti-VLP18 Ig antibody response measured on individualsera on day 14 post II

Similar anti-VLP18 titers were obtained after immunisation with thecombination of HBs and VLPs (group G and H) or with the monovalent VLPsformulations (group A) (GMT of 37285 or 51202 EU/ml versus 56504 EU/ml)Equivalent titers (group G and H) were observed between the combinationprepared using either ways to adsorb the 3D-MPL: Al(OH)₃ alone (group G)compared to mixed adsorption on Al(OH)₃ and AlPO₄ (group H).

These differences were shown as statistically not significant usingone-way analysis of variance test.

2. Cell Mediated Immune Response

Cell-mediated immune responses (lymphoproliferation, IFNγ/IL5production) were evaluated at day 14 post II after in vitrorestimulation of splenic cells with either HBs, E7 or VLPs antigens. Foreach group of mice, pools of 5 organs were constituted.

The experimental procedure is fully described above in Material andMethods.

3. Cytokine Production

a) In vitro Restimulation with HBs

FIG. 5 shows the cytokine production monitored in splenic cells after 96h in vitro restimulation with HBs.

Low IFN-γ and IL5 production was observed for all groups but as shown inTable 2 higher production of IFN-γ are observed compared to IL-5production with IFN-γ/IL-5 ratio indicating that a comparable TH1response is induced with the monovalent and combined vaccines. The groupC results should not be taken into account as data below the thresholdmay indicate absence of antigen for restimulation.

TABLE 2 IFN-γ/IL-5 ratio after in vitro restimulation with HBs. RatioIFN-γ/IL-5 Group C Group D Group E Group F Group G Group H HBs 10 μg/ml0.3 4.0 9.4 7.9 7.9 6.6 HBs 1 μg/ml 0.4 3.7 1.5 4.0 4.0 5.0b) In vitro Restimulation with E7

FIG. 6 shows the cytokine production monitored in splenic cells after 96h in vitro restimulation with E7 antigen.

A dose range effect was observed when comparing the 10 μg and 1 μg Agdose for restimulation.

A non-specific response was observed for HPV16/18 L1 VLPs immunisedgroups using 10 μg of Ag for restimulation.

The IFN-γ is produced in a much higher concentration compared to IL-5(Table 3) indicating a clear TH-1 profile of the immune response in allgroups evaluated (monovalent versus combination).

TABLE 3 IFN-γ/IL-5 ratio after in vitro restimulation with E7. RatioIFN/IL-5 Group B Group F E7 10 μg/ml 17.7 12.9 E7 1 μg/ml 8.9 1.2c) In vitro Restimulation with VLP16 and 18

FIGS. 7 and 8 show the lymphoproliferation after in vitro restimulationwith VLP16 or VLP18 on day 14 post II.

Comparable profiles were observed for all the formulations containingVLPs (Integrated Stimulated Indexes between 12-29) with cpm around 30000for 10 μg Ag restimulation dose, indicating the absence of interferencebetween the different formulations on this read-out.

FIG. 9 shows the cytokine production monitored in splenic cells after 96h in vitro restimulation with VLP16.

FIG. 10 shows the cytokine production monitored in splenic cells after96 h in vitro restimulation with VLP18.

No dose range effect has been observed using 10 μg and 1 μg Ag dose forrestimulation with either VLP antigens on both cytokine production.

A clear TH1 profile was observed with all formulations.

TABLE 5 IFN-γ/IL-5 ratio after in vitro restimulation with VLP16 andVLP18. Ratio IFN/IL-5 Group A Group G Group H VLP16 10 μg/ml 12.0 19.916.5 VLP16 1 μg/ml 22.1 37.9 23.2 VLP18 10 μg/ml 20.5 17.9 13.4 VLP18 1μg/ml 21.8 23.7 21.0

CONCLUSIONS

The effect of the combination of VLPs/HBs or E7/HBs Ag formulated inAS04 on the immunogenicity was evaluated in Balb/C mice:

Regarding the serological analysis, no interference of the Agcombination was observed on anti-HBs, anti-E7 and anti-VLPs serology.

The combination of VLPs and HBs or E7 and HBs antigens did not interferewith the isotypic profile of the antibody response displayed by themonovalent vaccine.

The method of adsorption of 3D-MPL (Al(OH)₃, AlPO₄, or mixtures ofAl(OH)₃ and AlPO₄) did not interfere with the serological results.

In the lymphoproliferation assays, results were available afterrestimulation with the VLPs. In these groups, no negative effect of thecombination of Ag was observed on the proliferative response.

For the cytokines evaluation, low cytokine production (IL-5 and IFN-γ)was obtained after restimulation with HBs Ag but responses werecomparable in the monovalent and combined vaccines. After restimulationwith E7 or with VLPs, comparable cytokine levels were producedrespectively in the E7/HBs or in the VLP/HBs combination as compared tomonovalent groups. The TH-1 profile observed with each monovalentvaccine was conserved in the combination vaccine groups.

1. A vaccine composition comprising: (a) a hepatitis B viral antigen;(b) an HPV-16 L1 antigen or HPV-16 VLP comprising an HPV-16 L1 antigen;and (c) an HPV-18 L1 antigen or HPV-18 VLP comprising an HPV-18 L1antigen; in conjunction with an adjuvant which is a preferentialstimulator of TH1 cell response wherein the vaccine compositionstimulates a TH1-type immune response.
 2. A vaccine compositionaccording to claim 1 which additionally comprises a carrier.
 3. Avaccine composition according to claim 1 in which the preferentialstimulator of TH1-cell response is selected from the group of adjuvantscomprising: 3D-MPL, 3D-MPL wherein the size of the particles of 3D-MPLis preferably about or less than 100 nm, QS21, a mixture of QS21 andcholesterol, and a CpG oligonucleotide.
 4. A vaccine compositionaccording to claim 3 in which the preferential stimulator of TH1-cellresponse is 3D-MPL.
 5. A vaccine composition according to claim 1 inwhich the Hepatitis B antigen is hepatitis B surface antigen.
 6. Avaccine composition consisting essentially of: (a) a hepatitis B viralantigen; (b) an HPV-16 L1 antigen or HPV-16 VLP comprising an HPV-16 L1antigen; and (c) an HPV-18 L1 antigen or HPV-18 VLP comprising an HPV-18L1 antigen; in conjunction with an adjuvant which is a preferentialstimulator of TH1 cell response wherein the vaccine compositionstimulates a TH1-type immune response.
 7. A vaccine compositionconsisting of: (a) a hepatitis B viral antigen; (b) an HPV-16 L1 antigenor HPV-16 VLP comprising an HPV-16 L1 antigen; (c) an HPV-18 L1 antigenor HPV-18 VLP comprising an HPV-18 L1 antigen; and (d) an aqueouscarrier in conjunction with an adjuvant which is a preferentialstimulator of TH1 cell response wherein the vaccine compositionstimulates a TH1-type immune response.